1. Field of the Invention
The present invention relates to the field of antisense technology. The present invention also relates to using the antisense technology in therapeutics and in gene function identification systems.
2. Description of the Background
Antisense molecules bind to complementary sequences of mRNA through Watson-Crick base pairing. Antisense oligonucleotides (AS-oligos) have been valuable in the functional study of a gene by reducing gene expression in sequence specific manner (Thompson et al. Nature, 314, 363-366 (1985); Melani et al. Cancer Res., 51, 2897-2901 (1991); Anfossi et al. Proc. Natl. Acad. Sci. USA, 86, 3379-3383 (1989)). Intense effort has also been made to develop antisense anticancer agents that eliminate aberrant expression of genes involved in tumor initiation and progression (Kamano et al. Leuk. Res., 14, 831-839 (1990); Melotti et al. Blood, 87, 2221-2234 (1996); Ferrari et al. Cell growth Differ., 1, 543-548 (1990); Ratajczak et al. Blood, 79, 1956-1961 (1992); Kastan et al. Blood, 74, 1517-1524 (1989); Thaler et al. Proc. Natl. Acad. Sci. USA, 93, 1352-1356 (1996); Wagner Nature, 372, 333-335 (1994)). Synthetic AS-oligos have been widely utilized for their ease of design and synthesis as well as potential specificity to a target gene. Antisense inhibition of gene expression is believed to be achieved either through RNaseH activity following the formation of antisense DNA-mRNA duplex or through steric hindrance of the mRNA movement to bind a ribosomal complex (Dolnick Cancer Invest., 9, 185-194 (1991)). There has also been an effort to inhibit gene expression by employing oligonucleotides that form triple helix aimed at the promoter region of the genomic DNA. Moreover, duplexed oligonucleotide decoys that compete with the promoter region of genomic DNA has also been formed (Young et al. Proc. Natl. Acad. Sci. USA, 88, 10023-10026 (1991)). Efficacy of AS-oligos has been validated in animal models as well as several recent clinical studies (Offensperger et al. EMBO J., 12, 1257-1262 (1993); Tomita et al. Hypertension, 26, 131-136 (1995); Nesterova et al. Nat. Med., 1, 528-533 (1995); Roush Science, 276, 1192-1193 (1997)). In addition, the first antisense drug was approved for CMV retinitis in US and Europe.
Expectations for AS-oligos have, however, frequently met with disappointment, as results have not always been unambiguous. Some of the problems of using AS-oligos have been inaccessibility to a target site (Flanagan et al. Mol. Cell Biochem., 172, 213-225 (1997); Matsuda et al. Mol. Biol. Cell, 7, 1095-1106 (1996)), instability to nucleases (Akhtar et al. Life Sci., 49, 1793-1801 (1991); Wagner et al. Science, 260, 1510-1513 (1993); Gryaznov et al. Nucleic Acids Res., 24, 1508-1514 (1996)), lack of sequence specificity and various side effects in vivo. The stability of AS-oligos has improved to a certain extent by using chemically modified oligos, which are the so-called second generation AS-oligos (Helene Eur J Cancer, 27(11),1466-71 (1991); Bayever et al. Antisense Res. Dev. 3(4), 383-90 (1993); Baker et al. Biochim. Biophys. Acta., 1489, 3-18 (1999)). Phosphorothioate (PS)- and methylphosphonate (MP)-oligos, have been exhaustively studied and are utilized mainly to augment stability to nucleases. However, each of the modified AS-oligos exhibit both lack of sequence specificity and insensitivity to RNaseH. Further, there has been concern over inadvertent introduction of mutations during DNA replication or repair caused by recycling of hydrolyzed modified nucleotides.
A series of distinct antisense molecules with enhanced stability, the so-called ‘third generation AS-oligos’, having 1) a stem-loop structure, 2) the CMAS (Covalently-closed Multiple Antisense) structure and 3) the RIAS (Ribbon Antisense) structure (Moon et al. Biochem J., 346, 295-303 (2000); Matsuda et al. Mol. Biol. Cell, 7, 1095-1106 (1996); Moon et al. J. Biol Chem., 275(7), 4647-53 (2000)) have been described. Both CMAS and RiAS-oligonucleotides exhibit enhanced stability to exonucleases and nucleases in biologic fluids. These antisense molecules are also efficacious in the specific reduction of target mRNA. However, there is a need in the art to develop an antisense molecule with greater facility and enhanced binding efficiency.
Certain bacteriophages, such as M13 bacteriophage, have a single stranded circular genome, which has been employed for DNA sequencing analyses as well as mutagenesis studies. M13 phagemid, which is a plasmid used in the construction of a recombinant bacteriophage, can be engineered to produce a large quantity of circular single stranded genomic DNA that contains an antisense sequence to a specific gene. This approach for producing antisense DNA takes advantage of the stability to exonucleases associated with the covalently closed structure, high sequence fidelity, elimination of laborious target site search and easy construction of an antisense library.
Tumor Necrosis Factor alpha (TNF-α) is a cytokine required for normal immune response (Perkins et al. Arthritis Rheum., 41(12), 2205-10 (1998)), septic shock (Camenisch et al. J. Immunol., 162(6), 3498-503 (1999)), and graft versus host response when produced in excess (Hill et al. J. Immunol., 164(2), 656-63 (2000)). TNF-α is also a typical proinflammatory cytokine and is closely linked with rheumatoid arthritis, allergy and other immunological disorders including sepsis and inflammatory conditions (Perkins et al. Arthritis Rheum., 41(12), 2205-10 (1998)). TNF-α expression can be induced in rat monocytic cell line WRT7/P2 by lipopolysaccharide (LPS) treatment.
Nuclear factor-κB (NF-κB) regulates a variety of genes for cytokines (Collins Lab Invest., 68, 499-508 (1993)); Collins et al. FASEB J., 9, 899-909 (1995) and adhesion receptors (Thanos et al. Cell, 80, 529-532 (1995)). Activated NF-κB was identified in macrophages, smooth muscle cells and endothelial cells of human artherosclerotic tissue (Defillipi et al. Curr Top Microbiol Immunol., 184, 87-98 (1993)) suggesting an important role of NF-κB in inflammatory and proliferative processes (Brand et al. J Clin Invest., 97, 1715-1722 (1996)).
The ras oncogene is frequently activated in human neoplasms. RAS p21 proteins are a part of the large family of GTP/GDP-binding proteins, located on the inner side of the plasma membrane. A point mutation in the ras gene can create a RAS p21 protein that fails to hydrolyze its bound GTP and thus transmits an intracellular signal resulting in unregulated cell proliferation (Alberts et al. Molecular biology of the cell. Garland, New York, 1273-1290 (1994)).
The protooncogene c-myb plays an important role in the proliferation and differentiation of hematopoietic cells. Hematopoietic cells show differential expression of c-myb and exhibit little expression of the gene upon terminal differentiation (Melotti et al. Blood, 87, 2221-2234 (1996); Ferrari et al. Cell growth Differ., 1, 543-548 (1990)). The c-myb gene product has been frequently found to be overexpressed in leukemic cells (Melani et al. Cancer Res., 51, 2897-2901 (1991); Anfossi et al. Proc. Natl. Acad. Sci. USA, 86, 3379-3383 (1989); Kamano et al. Leuk. Res., 14, 831-839 (1990)).
The MYC oncoproteins play a central role in tumor cell growth (Packham et al. Biochim. Biophys. Acta., 1242, 11-28 (1995); Henriksson et al. Adv. Cancer Res., 68, 109-182 (1996)). MYC is sufficient to induce quiescent cells to enter the cell cycle (Eilers et al. Nature, 340, 66-68 (1989)), suggesting that it is required for continuous cell growth, while its inhibition can intervene in mitogenic signaling and induce cells to differentiate terminally (Heikkila et al. Nature, 328, 445-448 (1987); Sawyers et al. Cell, 70, 901-910 (1992)).
The principal components of the cell cycle regulatory genes are represented by a family of protein kinases termed cyclin-dependent kinases (CDKs). A greater understanding of molecular events controlling the transition from one phase of cell cycle to the next has engendered the discovery of CDKs (Morgan Nature (Lond.), 374, 131-134 (1995)). CDKs are inactive until they bind to their coactivators, which are individual cyclin proteins. As cells enter G1, kinase activities of CDK4 and CDK6 appear necessary for transition through early G1 checkpoints (Sherr Cell, 73, 1059-1065 (1993)), and the activity of the CDK2-cyclin E complex is necessary for transition from G1 into S phase (Ohtsubo et al. Mol. Cell. Biol., 15, 2612-2624 (1994)). The development of potent antisense oligonucleotides for CDK activity would represent an attractive approach for the inhibition of tumor cell growth.
There is a need in the art for more specific, stable and potent antisense molecules to be used against various human diseases.